Characterization and stress-responsive regulation of CmPHT1 genes involved in phosphate uptake and transport in Melon (Cucumis melo L.)

Background Phosphorus (P) deficiency, a major nutrient stress, greatly hinders plant growth. Phosphate (Pi) uptake in plant roots relies on PHT1 family transporters. However, melon (Cucumis melo L.) lacks comprehensive identification and characterization of PHT1 genes, particularly their response patterns under diverse stresses. Results This study identified and analyzed seven putative CmPHT1 genes on chromosomes 3, 4, 5, 6, and 7 using the melon genome. Phylogenetic analysis revealed shared motifs, domain compositions, and evolutionary relationships among genes with close histories. Exon number varied from 1 to 3. Collinearity analysis suggested segmental and tandem duplications as the primary mechanisms for CmPHT1 gene family expansion. CmPHT1;4 and CmPHT1;5 emerged as a tandemly duplicated pair. Analysis of cis-elements in CmPHT1 promoters identified 14 functional categories, including putative PHR1-binding sites (P1BS) in CmPHT1;4, CmPHT1;6, and CmPHT1;7. We identified that three WRKY transcription factors regulated CmPHT1;5 expression by binding to its W-box element. Notably, CmPHT1 promoters harbored cis-elements responsive to hormones and abiotic factors. Different stresses regulated CmPHT1 expression differently, suggesting that the adjusted expression patterns might contribute to plant adaptation. Conclusions This study unveils the characteristics, evolutionary diversity, and stress responsiveness of CmPHT1 genes in melon. These findings lay the foundation for in-depth investigations into their functional mechanisms in Cucurbitaceae crops. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-05405-w.


Background
Phosphorus (P) is an essential element of phospholipids and nucleic acids, which plays a crucial role in energy transfer reactions and signal transduction processes that are vital for all life forms on Earth [1].Although soils generally contain abundant P, only a small fraction is accessible for crop use [2].It is bound to incompletely weathered mineral particles, adsorbed on mineral surfaces, or forms occluded P through secondary mineralization [2].Therefore, roots obtain phosphate (Pi) against a huge concentration gap between plant cells (mM) and soil (µM) [3,4].So, efficient Pi acquisition from soil and translocation within plants are necessary to maintain general levels of cellular Pi [5].Root cell absorption of Pi from the soil is energy-dependent.The PHT1 family of plant Pi transporters are known to be the primary facilitator of this process [6,7].
Commonly, PHT1 family members are highly conserved among the different species [19].36 TaPHT1s have been found in wheat (Triticum aestivum), among which TaPHT1.1/1.9, 1.2, and 1.10 were expressed especially in roots [20].There are 14 PHT1s in soybean (Glycine max (L.) Merr.), which are expressed differently not only in responding to Pi availability but also in other nutrient deficiencies, including N, K, and Fe, in the different tissues [21].8 PHT1s showed diverse roles and genetic redundancy responding to Pi deficiency in tomato [22].Genome-wide analysis has identified 12 PHT1s in Gossypium hirsutum [23].9 CsPHT1s were identified in tea plants (Camellia sinensis L. O. kuntze), which play crucial roles in selenite homeostasis [24].201 PHT1 homologs were identified and analyzed from three diploids and two allotetraploids Brassica species, which were induced by heavy metal stress [25].However, the characterization of PHT1 gene family and the stressresponsive patterns are still unknown in melon.
Melon is an important crop for its unique flavor and nutritional value worldwide [26].Pi uptake and transport mediated by PHT1 play an important role in melon plant growth, fruit growth and quality formation, especially sugar accumulation.Furthermore, current melon cultivars have a narrow genetic base and are vulnerable to both biotic and abiotic stresses due to prolonged domestication and artificial selection for high yields and desirable traits [27,28].Therefore, breeders are facing the arduous work of improving melon resistance with conventional and/or modern breeding approaches.Our previous research revealed that three CmPHT1s positively participated in Pi uptake under Pi-deficiency [29].We speculated that there should be other PHT1s in melon.Are CmPHT1s involved in the other stress responses in melon?Further, what factors regulate the transcription of these PHT1 genes?In the current study, 7 CmPHT1 genes were identified and systematically analyzed based on the updated melon genome and their characteristics were analyzed including gene structure, conversed motif structure, chromosomal location, evolutionary relationship, synteny relationship, and cis-elements in the promoters.14 cis-element categories were identified in CmPHT1 promoters.The stress-responsive expression patterns of CmPHT1s in melon were surveyed under low-phosphate stress (LP) and other stresses.Combining the bioinformatical analysis, three CmWRKYs regulated CmPHT1;5 expression by binding to its W-box element.This research shed light on the characterization and stress-regulatory elements of CmPHT1s, which contribute to clarify further the functional mechanisms under biotic and abiotic stress in melon.

CmPHT1s were identified in melon
The BLAST search was used to identify all possible PHT1 members in melon genome.After the redundant and disrelated genes were deleted, and the conserved domains were ensured, 7 CmPHT1s were identified (Table S1).They were named CmPHT1;1 to CmPHT1;7 according to their chromosomal location.The CmPHT1 protein sequences exhibited high identity and similarity levels (Table S2).The identity of protein sequences ranged from 47 to 80%.The similarities ranged from 62 to 89%.The highest identity and similarity were found between the protein sequences of CmPHT1;3 and CmPHT1;5.
The basic characteristics were analyzed (Table S1).These CmPHT1s ranged from 519 (CmPHT1;4) to 556 (CmPHT1;7) amino acids in size, with a molecular weight of around 56 to 61 kDa.PI values were from 8.57 (CmPHT1;4) to 9.07 (CmPHT1;1).The proteins with II value over 40 are unstable and those below 40 are stable proteins [30].All CmPHT1 proteins are stable proteins.AI of 7 CmPHT1 proteins ranged from 88.08 (CmPHT1;2) to 94.68 (CmPHT1;5).The sub-cellular localization of the CmPHT1s was predicted using protein localization prediction software.The consistent results revealed that all CmPHT1 proteins were localized in the plasma membrane.These transporters shared a similar topology with 12 membrane-spanning domains.

Phylogenetic analysis of the CmPHT1 proteins
To predict the phylogenetic relationship and function of CmPHT1 proteins, a phylogenetic tree was constructed using the full-length PHT1 protein sequences of melon, Arabidopsis, and rice (Fig. 2).The CmPHT1s were clustered into five clusters (C1, C2, C3, C4, and C5).The phylogenetic analysis revealed that each gene from melon exhibited the closest relationship with corresponding genes from Arabidopsis.The following pairs were yielded: CmPHT1;2/1;4 and AtPHT1;4, CmPHT1;3/1;5

Expression profiling of CmPHT1s with RNA-seq under pathogen infections
To examine responses of the CmPHT1 genes to biotic stress, the transcriptome data were extracted for CmPHT1 gene expression at 0 (CK), 24, 72, and 168 h post-inoculation (hpi) by Podosphaera xanthii (Px) which caused the powdery mildew (PM) in the two contrasting cultivars (the resistant 'MR-1' and the susceptible 'Topmark') [31], at 0 (control, CK),3 and 5 d post-inoculation (dpi) by Phytophthora capsica in the tolerant line 'L8' Fig. 3 Illustrative depiction of the chromosomal distribution and interchromosomal relationships of CmPHT1s.Gray lines, red lines, and green genes indicated all synteny blocks, duplicated CmPHT1 gene pairs, and tandem duplication of CmPHT1s in the melon genome, respectively [32].CmPHT1;3 was up-regulated at 168 hpi, while there was no difference at 24 and 72 hpi in resistant leaves (Fig. 5A).CmPHT1;4 was up-regulated during the whole Px infection in resistant leaves (Fig. 5A).There was no CmPHT1 gene responding to Px infection in the susceptible melon genotype.It suggested that CmPHT1;3 and CmPHT1;4 involved in the Pi transport to leaves infected by Px in resistant melon genotype.
The global CmPHT1s of the resistant and susceptible genotypes at 3 and 5 dpi compared with CK in roots were analyzed (Fig. 5B).CmPHT1;3, CmPHT1;4, and CmPHT1;5 were up-regulated at 5 dpi in the resistant genotype.CmPHT1;3 and CmPHT1;5 were downregulated in the susceptible genotype compared with CK.Phytophthora capsici inhibited the expression of CmPHT1;3 and CmPHT1;5 in the susceptible genotype roots.It indicated that CmPHT1;3, CmPHT1;4, and CmPHT1;5 played a positive role by promoting the uptake of Pi in resistance to P. capsici in the melon root.

Expression profiling of CmPHT1s with RNA-seq under cadmium stress
The transcriptome data under cadmium (Cd) stress in the melon root were analyzed to check CmPHT1 expression [33].The expression of CmPHT1s in melon root was suppressed by Cd and not improved by pretreated with 1 µmmol L − 1 GR24 solution (Fig. 6A).It indicated that Cd inhibited the expression of CmPHT1s and the Pi absorption by root from the cultivation medium.It may be one way of growth inhibition for melon seedlings by Cd.

Expression profiling of CmPHT1s with RNA-seq under waterlogging stress
The CmPHT1s were identified as participating in the development of adventitious roots induced in melon with the transcriptome profiling data under waterlogging [34].CmPHT1;5 was significantly up-regulated during waterlogging, and CmPHT1;7 was significantly up-regulated at 72 HAW (Fig. 6B).Interestingly, it was corresponding to the AREs in the promoters of CmPHT1;5 and CmPHT1;7 (Fig. 4).Waterlogging might induce some factors to bind the ARE elements and activate the expression of CmPHT1;5 and CmPHT1;7.

Expression profiling of CmPHT1s with RNA-seq under the low-phosphate stress (LP) in two contrasting melon genotypes
The growth status of the tolerant cultivar '46 − 2' was better than sensitive cultivar '26 − 1' under LP (Fig. 7A).The P contents of root, stem, and leaves, except the second and third leaves in '46 − 2' were significantly higher than '26 − 1' (Fig. 7B).CmPHT1;3 at 0 and 0.5 d and CmPHT1;7 at 7 and 14 d after LP were significantly upregulated in the sensitive cultivar compared with the tolerant cultivar.It indicated that CmPHT1;3 was rapidly induced in the sensitive cultivar (Fig. 7C).No differences in the expression of CmPHT1;3 were found after 2 d.The roots mobilized the CmPHT1s to absorb more P in the LP-sensitive cultivar, which also reflected that the P content was less in the sensitive cultivar.Combined with the cis-elements, LP might induce the expression of PHR1 and then activate CmPHT1;7.

Expression patterns of CmPHT1s under the hightemperature stress
To explore the expression response to high temperature, we measured the transcript level of CmPHT1s (Fig. 8).The expression of CmPHT1;3, CmPHT1;6, and CmPHT1;7 initially surged, peaking at the 1st h, and subsequently declined in leaves subjected to high-temperature stress (HT) (Fig. 8).CmPHT1;4 was downregulated by HT in leaves (Fig. 8), while there was no effect on the transcript level of CmPHT1;5 in leaves (Fig. 8).The transcript level of CmPHT1;3, CmPHT1;4 and CmPHT1;7 decreased markedly at 1st h, increased at 3rd h, and peaked at the 12th h in stems.The transcript of CmPHT1;3 and CmPHT1;7 in roots decreased under  HT.While the expression of CmPHT1;4 and CmPHT1;5 decreased firstly at 1st h, and then showed an increasing trend subsequently in roots.

Expression of CmPHT1s under the low-nitrate stress
The CmPHT1;3 expression was downregulated in leaves and stems under the low-nitrate stress (LN) (Fig. 9).CmPHT1;3 expression was downregulated at 1st -3rd h and upregulated at 6th h in roots.CmPHT1;4 was downregulated in leaves, stems, and roots by LN.CmPHT1;5 was downregulated in leaves and roots by LN, while upregulated at 3rd and 6th h in stems.CmPHT1;7 was downregulated in leaves, stems, and roots by LN.As a whole, LN suppressed the expression of CmPHT1s.

Expression patterns of CmPHT1s under the low-phosphate stress
The CmPHT1s expression was assessed in the LP-tolerant cultivar (Fig. 10).CmPHT1;3 was downregulated in leaves and stems under LP.It was upregulated at 24th h in roots.CmPHT1;4 was upregulated in leaves at the 6th and 12th h and downregulated in stems and roots by LP.CmPHT1;5 was upregulated in leaves at the 6th and 24th h, in stems at 1st and 3rd h, and downregulated in roots by LP.CmPHT1;7 was downregulated in leaves, stems, and roots by LP, except the 1st h and 24th h in leaves.
CmWRKYs directly bound to the W-box element in CmPHT1;5 promoter CmPHT1;5 was upregulated, and three CmWRKY genes (CmWRKY31, CmWRKY41, and CmWRKY18) were upregulated under stresses by analyzing the RNA-seq data (Table S4).Further, we analyzed the promoter sequences and found that a W-box element existed from − 699 bp to -693 bp in CmPHT1;5 promoter (Figs. 4 and  11A).It was assumed that CmWRKY31, CmWRKY41, and CmWRKY18 may be the upstream transcription factor (TF) of CmPHT1;5.To verify this hypothesis, a yeast-one-hybrid (Y1H) assay was performed with CmWRKYs.All yeast cells grew well on SD/-Ura/-Trp  medium; however, the yeast strain EGY48 blued only when co-transformed CmWRKY31, CmWRKY41or CmWRKY18 and the CmPHT1;5 promoters (Fig. 11B).It showed that pB42AD-CmWRKYs fusion protein strongly activated the expression of LacZ.In addition, we identified that WRKY recognition sites were W-box element (TTGACC) by Y1H (Fig. 11C).The results demonstrated that CmWRKYs are directly bound to the W-box element in CmPHT1;5 promoter.

Discussion
In recent years, numerous PHT1 gene families have been discovered in diverse plant species employing comparative genome methodologies.The identification of seven putative PHT1s in the melon genome came true through extensive bioinformatics analysis.Studying the gene structure, promoter region cis-elements, evolutionary relationship, chromosomal distribution, and expression profiles of CmPHT1s can offer insights into the mechanisms underlying the conservation, expansion, and functional diversity of PHT1s across the entire Cucurbitaceae family, facilitating a comprehensive understanding of their potential roles.
CmPHT1s were inhibited by LN in melon roots (Fig. 9).It has been reported that N availability regulates Pi-deficiency responses [43].Under P-deficiency, N supplement is conducive to P acquisition, while N-starvation restrains the P-starvation responses, for example the expression of PHT1 genes [44].A downregulation of PSR (Phosphate starvation response) genes emerges in rice and maize under N starvation [45,46].Three major signaling factors SPXs, PHR, and PHO2 have been involved in N-P interaction.It has been reported that PHR is positively regulated by N at transcriptional and post-transcriptional levels [47,48].However, the stability of PHR1 decreases under N-starvation [49].
After pathogen infection, CmPHT1s exhibited upregulation in the leaves of the resistant cultivar and downregulation in the leaves of the susceptible cultivar (Fig. 5).It indicated that the pathogen inhibited the expression of CmPHT1s in the susceptible cultivar leaves.CmPHT1;3 and CmPHT1;4 can participate in the transport of Pi into the leaves.It has been reported that the proportion of the total phosphate in shoots was more in mildewed than in healthy barley [53].Our previous research found that CmPHT1s were upregulated in the powdery mildew (PM)-resistant cultivar compared with the PM-susceptible cultivar under LP-stress (not published).Foliar applications of mono-potassium phosphate fertilizer inhibit powdery mildew development in nectarine trees [54].The foliar sprays of phosphate and potassium salts can control PM caused by Sphaerotheca fuliginea in cucumber [55].The OsPT8-overexpressed rice plants compromised the resistance against M. oryzae and X. oryzae pv.Oryzae.It indicates that the cross-talk between OsPT8, Pi signaling, and plant immunity exists [56].The excess of Pi enhances disease susceptibility to M. oryzae in rice.It indicated that Pi and defense signals must operate in a coordinated manner to control disease resistance [57].
Even though CmPHT1s aren't the pivotal genes that regulate the defense against the pathogen, they could positively participate in the resistance to PM in melon.
Thus, the transcript of CmPHT1s is differentially regulated by stresses.The expression profiles of PHT1s could be integral to stress adaptation mechanisms.Nonetheless, the regulatory mechanisms remain to be studied further.

Cis-elements in CmPHT1 Promoters revealed their functional divergence
The growth and development of melons are susceptible to a variety of biotic and abiotic stresses.The stresses such as pathogen infection, nutrition deficiency, heavy metal pollutants, waterlogging, and extreme temperature often have injurious effects on melon growth and development [31][32][33][34][35].The cis-regulatory elements in the CmPHT1 promoter regions are the premise of CmPHT1 functions [58].Most CmPHT1 promoters contained abiotic stress-responsive elements, suggesting that CmPHT1 genes widely participate in stress responses.P1BS to which PHR1 binds is a conserved motif responding to phosphate deficiency in crops [59].CmPHT1;4, CmPHT1;6, and CmPHT1;7 possess P1BS in the promoter regions (Fig. 4).CmPHT1;6 was not induced by Pi deficiency, although it contained P1BS elements (Figs. 4  and 10).It was consistent with the previous conclusions [60,61].There were 4 AREs in the promoter regions of CmPHT1;5 (Fig. 4).CmPHT1;5 was significantly upregulated under waterlogging stress (Fig. 6B).It indicated that CmPHT1;5 participated in the response to waterlogging in melon roots.Each CmPHT1 gene has plenty of lightresponsive cis-elements (Fig. 4).It has reported that red light regulated the transcript of PHT1;1 by binding two PHYTOCHROME-INTERACTINGFACTORs and ELONGATED HYPOCOTYL 5 to the PHT1;1 promoter [62].It suggests that PHT1s involve positively in the crop growth and development.
The cis-elements, such as methyl jasmonate, gibberellin, salicylic acid, auxin and abscisic acid response, exist in the CmPHT1 promoters.It implies that CmPHT1s are regulated by hormone signals.The regulation of PHT1s is affected by phytohormones that mediate stress signals, such as ethylene and abscisic acid, as well as other phytohormones like auxin, cytokinin, and gibberellin [63].In Arabidopsis, the expression of AtPHT1;1 was significantly reduced by cytokinin and increased by auxin [64].The transcript of Pht1;1 was induced by auxin and GA in maize [65].Further study on the regulatory mechanism of phytohormones to CmPHT1s needs to be done.

CmWRKYs are the upstream TFs of CmPHT1;5
Of the 7 CmPHT1s, CmPHT1;5 was chosen for the in-depth function analysis based on the following reason: first, CmPHT1;5 was widely responsive to stresses (Figs.5B, 6B and C, 8, 9 and 10), indicating its potential broader functions.Second, based on the bioinformatical analysis of its promoter, we noticed that it contains various cis-regulatory elements associated with stress responsiveness (anaerobic induction, MYB binding site, low-temperature response, WRKY binding site, defense and stress response), growth and development (light response and meristem expression), metabolic regulation (zein metabolism regulation), and hormone responsiveness (salicylic acid response and gibberellin response) (Fig. 4), indicating that it may be regulated not only by stress but also growth and development.Thirdly, CmPHT1;5 is not only responsible for the absorption of Pi, but also for its transport from root to shoot.All the evidence strongly supports CmPHT1;5 as a promising candidate gene for in-depth functional characterization in future study.
WRKY TF family members play key roles in regulating plant Pi homeostasis.Based on the transcriptional data published in melon, CmWRKY41, CmWRKY31, and CmWRKY18 shared the same expression patterns with CmPHT1;5 (Table S4).Y1H assay results showed three CmWRKYs directly bound to the W-box element in its promoter (Fig. 11).It indicated that CmWRKYs are the upstream TFs of CmPHT1;5.AtWRKY45 directly upregulates of AtPHT1;1 expression by binding to two W-boxes within the AtPHT1;1 promoter under Pi starvation [66].OsWRKY21 and OsWRKY108 function redundantly to promote Pi uptake by activating OsPHT1;1 expression under Pi-replete condition [67].OsWRKY74 significantly influenced Pi acquisition by regulating OsPHT1;3, OsPHT1;4, and OsPHT1;10 transporter proteins in OsWRKY74-overexpressed lines [68].Under the abundant Pi, on one hand, PtoWRKY40 binds to the W-box of PtoPHT1s promoter to repress their expression; on the other hand, PtoWRKY40 interacts with PtoPHR1-LIKE3 (PtoPHL3) to prevent PtoPHL3 from binding to the P1BS of PtoPHT1s and thus reduced PtoPHT1s' expression.However, Pi deficiency decreased PtoWRKY40 abundance and therefore initiates PtoPHT1s' expression [69].These reports indicated that PHT1s are the target genes of WRKY TFs under the different Pi conditions.These works provide the ideas and methods for future studies.However, many further works need to be done in melon, for example, CmPHT1 function, the CmWRKY-regulatory mechanism, and the stress-regulatory mechanism to CmPHT1s expression.
CmPHT1s played the positive roles in stress tolerance, and some TFs regulated the CmPHT1expression.Modern breeding methods, such as transgenesis and gene editing technique, are effective and trusted in improving crop stress tolerance.Previous reports have shown that overexpression of PHT1s or their upstream TFs in crop improved the tolerance to stresses.Overexpression of OsWRKY74 significantly enhanced tolerance to Pi starvation in rice [68].OsPHT1;3 overexpression led to increased Pi concentration in rice by improving the absorption and transport of Pi under extremely LP regimes [12].EsPHT1;5 overexpression in salt cress enhanced plant tolerance to LP and salinity by playing an integral role in Pi acquisition and distribution [70].These findings indicated that PHT1s and their upstream TFs can serve as targets for genetic manipulation to improving crop tolerance to stresses.We can transfer CmPHT1s or their positive regulatory TFs into melon and obtain the CmPHT1s overexpression plants.The plants will improve the Pi absorption and/or transport to meet Pi demand for stress resistance.
This study on CmPHT1s has many limitations for lack of function validation.The intensive exploration of their biological function and upstream TF identification should be carried out.For example, CmPTH1s are positively or negatively regulated by CmWRKYs, and the crucial function of CmPTH1s and their upstream TFs in melon.Moreover, the hormone-regulatory mechanism for CmPHT1s expression, for example auxin, abscisic acid, jasmonate, salicylic acid, and gibberellin, should be clarified under the stresses.Therefore, transgenic melon via overexpressing or/and knocking out the CmPTH1s and their upstream TF genes should be obtained, their physiologic functions response to various stresses and the regulatory mechanism need to be demonstrated in future research.

Conclusions
Our work identified 7 CmPHT1 genes in the melon genome.A comprehensive bioinformatic analysis of the CmPHT1 gene family was conducted, including basic characteristics, conserved domains, phylogenetic relationships, exon-intron structures, and promoter cis-elements.By combining expression pattern analysis with promoter studies, CmPHT1s were shown to respond to a variety of stresses, including phosphate deficiency, heat, anaerobic conditions, and pathogen infection.Interestingly, CmPHT1;3 and CmPHT1;5 displayed the most significant upregulation in response to these stresses.Future, CmWRKYs regulated the CmPHT1;5 expression by binding to the W-box element.In conclusion, the candidate CmPHT1s responsive to stresses were screened, laying some foundations for penetrative studies on their functional mechanism in Cucurbitaceae crops.

Plant materials, growth conditions, and stress treatments Low-phosphate stress
To investigate the response of CmPHT1 genes to LP stress in melon, two contrasting melon genotypes '46 − 2' (LP-tolerant genotype) and '26 − 1' (LP-sensitive genotype) were grown in the greenhouse facilities of Shanghai Jiao Tong University.These two genotypes were collected and saved by our group.The LP treatments were carried out under hydroponic conditions.Standardized seedlings with fully developed first true leaves were transferred to plastic trays, with each tray accommodating 18 plants.These trays were filled with 6 L of half-strength modified Hoagland's nutrient solution.The dose for LP treatment was 0.001 mM KH 2 PO 4 and the control (CK) was 0.25 mM KH 2 PO 4 based on the previous research [71].The plants were cultured at 28 °C/18 °C day/night with a 14-hour (h) photoperiod, 600 ± 20 µmol m − 2 s − 1 irradiance, and 50-75% relative humidity.LP treatment lasted for 21 days (d).
High-temperature treatment '46 − 2' (LP-tolerant genotype) was used in high-temperature treatment.The high temperature was maintained at 45 °C during the day and 35 °C during the night, with a 14-hour photoperiod, for one day [72].For control treatments, the temperature was 28℃/18℃ for day and night (14 h/10 h).

Low-nitrate treatment
'46 − 2' (LP-tolerant genotype) was used in low-nitrate treatment.The nitrate concentration of low-nitrate stress was 1% of control (8 mM) for one day [45].The culture condition was same with LP stress.
Samples were collected at 0 h, 1 h, 3 h, 6 h,12 h, 24 h, 2 d, 4 d, 7 d, 14 d, and 21 d post-treatment.These samples were harvested to assess various biochemical, physiological, and morphological parameters, with at least three subsamples taken for each measurement.

Download of data resources
The latest genome sequence and annotation files of melon (DHL92 V4.0) were downloaded from CuGenDB (http://cucurbitgenomics.org/organism/20) to construct a local database.The genome data of Arabidopsis and rice were downloaded from the ensemble database (http:// plants.ensembl.org/index.html) to analyze all the candidate PHT1 genes and construct the phylogenetic tree.

Genome-wide identification of CmPHT1s in melon
To get the comprehensive and accurate identification of CmPHT1s in melon genome, the following methods were taken.Firstly, 9 Arabidopsis PHT1 proteins (https:// www.arabidopsis.org/)and 13 OsPHT1 proteins (https:// rapdb.dna.affrc.go.jp/) were queried to search CmPHT1 proteins across the melon genome with 'Blast Compare Two Seqs (Sets)' in TBtools software [73].Secondly, the accuracy of deduced protein sequences was confirmed by searches for homologous sequences deposited in the NCBI database (https://www.ncbi.nlm.nih.gov/cdd/?term=) using the BLAST (Basic Local Alignment Search Tool) with E-values < 10 -5 .Thirdly, the candidate protein sequences were obtained and identified using the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/cdd/)(E-value < 10 5 , other parameters set as defaults) and SMART databases (http://smart.embl-heidelberg.de/) to ensure the presence of sugar (and other) transporter domains.The PHT1 genes that contained the transporter domain and the hits of E-values < 10 5 were considered CmPHT1 genes [74].The confirmed CmPHT1 genes were renamed according to their positions on melon chromosomes.

Phylogenetic tree construction
The full-length amino acid sequences of 7 CmPHT1, 9 AtPHT1, and 13 OsPHT1 proteins were made the alignment using ClustalW with default settings in MEGA 7 [75].An unrooted phylogenetic tree was constructed based on the alignments with the maximum likelihood (ML) method and 1000 bootstraps [76].For better visualization, the phylogenetic tree was beautified and embellished using the online tool Evolview v2 (https://www.evolgenius.info/evolview).

Analysis and visualization of gene structure and conserved motifs
The intron-exon distributions of CmPHT1s were obtained using GFF annotation files of melon genome.The gene structures of CmPHT1s were analyzed and visualized using Graphics of TBtools software [73].The conserved motifs were identified within the CmPHT1 proteins with MEME online.The ideal breadth of each motif was set to range from 6 to 50 residues.The number of motifs to find was set to 8.

Collinearity relationship
The genomic sequences and annotation data were scrutinized to extract collinearity data concerning the CmPHT1s using the TBtools.Subsequently, the results were imported into the Advanced Circos feature of TBtools for the visualization of expansion patterns within the CmPHT1 gene family.

Expression patterns of CmPHT1s under diverse stresses
Transcriptome data accessible online were employed to investigate the expression dynamics of CmPHT1s in melon under both biotic and abiotic stress conditions [31][32][33][34][35].The expression data of CmPHT1s were searched, analyzed, and visualized.

qRT-PCR analysis
The expression patterns of CmPHT1 family genes were analyzed via qRT-PCR.RNA was extracted using a plant RNA isolation reagent (Tiangen Biotech, China) and subjected to reverse transcriptase reactions to synthesize cDNA using PrimeScriptTM RT Master Mix.qRT-PCR was performed on a Roche LightCycler 96 real-time PCR machine (Roche, Basel, Switzerland) with four replicates.The calculation of the expression level was conducted as the relative 2 −△△Ct method [78].Actin was employed as an internal control [29,72,79].The primers are listed in Table S5.

One-hybrid (Y1H) assay
The genomic DNA was extracted from melon with a Plant Genomic DNA Extract Kit (Beijing Tiangen, China).The CmPHT1;5 promoter sequence was cloned according to the melon genomic sequence.

Statistical analysis
All experimental data were presented as the mean ± SE of at least three biological replicates.ANOVA analysis at P < 0.05 was performed to identify significant differences using SPSS Statistics 22.0 (IBM, Chicago, IL, USA).All figures were drawn with TBtools V1.120 (Guangzhou, China) and OriginPro 2022 (OriginLab, Northampton, MA, USA).

Fig. 1
Fig. 1 The phylogenetic tree, conserved protein motifs, conserved domain, and gene structure of CmPHT1s.(A) the phylogenetic tree; (B) conserved motifs in the CmPHT1 proteins.Each motif was represented by a colored box.(C) conserved domain; (D) gene structures of CmPHT1s.A yellow box, the black lines, and a green box represented the exons, introns, and UTR, respectively

Fig. 2
Fig. 2 Evolutionary relationships of PHT1 proteins between melon, Arabidopsis, and rice.The tree was generated with the maximum likelihood method with 1000 bootstrap replicates based on multiple alignments of amino acid sequences of 7 CmPHT1s, 9 AtPHT1s, and 13 OsPHT1s.The different colored backgrounds represented clusters of CmPHT1s.

Fig. 5
Fig. 5 Expression of CmPHT1s in melon under the different pathogen infections.The log 2 (foldchange of pathogen infection/CK) values were shown.The genes with thresholds of fold change (FC) ≥ 1.5 and false discovery rate (FDR) < 0.05 were identified as DEGs (same hereinafter).(A) the leaf infected by Podosphaera xanthii (Px) in the resistant 'MR-1' melon genotypes.T0, T24, T72, and T168 represented the hours post-inoculation.(B) the roots infected by Phytophthora capsici in melon.R: resistant cultivar, S: susceptible cultivar.0, 3, and 5 represented the day post-inoculation

Fig. 9
Fig. 9 Expression patterns of CmPHT1s under the low nitrate stress in melon.Significant differences within the same tissue across different treatment times are indicated by lowercase letters (P < 0.05).L: leaf; S: stem; R: root

Fig. 11 Fig. 10
Fig. 11 CmWRKYs bind to CmPHT1;5 promoter.(A) Schematic diagram of CmPHT1;5 promoter.The orange triangle represents the sequence of the W-box element in the CmPHT1;5 promoter and its relative positions to ATG.The predicted WRKY-binding site is located from − 699 to-693.(B) Y1H displays 3 CmWRKYs can interact with CmPHT1;5 promoter; (C) Three tandem copies of W-box element (TTGACC) were synthesized and ligated into pLacZ vector for Y1H assays